Ethoxyformylation of histidine residues in equine growth hormone

Reactivity of histidine residues in equine growth hormone to ethoxyformic anhydride was studied. The existence of two kinetically different sets was demonstrated: one of them including only the slow reacting histidine 169 (k = 0.164 min-1) and the other containing fast reacting histidines 19 and 21 (k = 0.892 min-1). A correlation between the decrease in the capacity to compete with 125I-labeled hormone for rat liver binding sites and the degree of ethoxyformylation of the fast group was found. Circular dichroism studies indicated no significant conformational changes in the protein with all three residues modified. These results fully agree with those obtained for bovine growth hormone which is further evidence supporting the vinculation of histidines 19 and/or 21 with the binding site of these hormones to their specific receptors.     

A novel proenkephalin processing carboxypeptidase and its activation by cyclic AMP dependent protein kinase

Carboxypeptidase B-like enzymes cleaving Met-enkephalin-Arg from synaptosomes of the rat striatum purified using a DEAE-cellulose column and Met-Arg-CH-Sepharose 4B affinity column proved to be different from enkephalin-convertase, lysosomal carboxypeptidase B-like enzyme, pancreas carboxypeptidase B and carboxypeptidase Y, in effects of inhibitors and activators, pH optimum (7.5-8.5) and molecular size (50,000). This enzyme, named "Processin CP-E" was activated by cAMP dependent protein kinase, and the Vmax was increased from 4.3 to 13.3 microM/min/mg protein, while the Km (28.2 microM) was unchanged.     

Conformational study of poly(L-lysine) interacting with acidic phospholipid vesicles

Circular dichroism measurements were carried out on poly(L-lysine) in the presence of vesicles of the negatively charged phospholipids, phosphatidylserine (PS; from bovine brain), phosphatidic acid (PA; prepared from egg yolk lecithin) and dimyristoylphosphatidylglycerol (DMPG). PS vesicles induced a conformational change in poly(L-lysine) from random coil to alpha-helix structure in 5 mM Tes (pH 7.0), whereas PA vesicles gave rise to beta-structure in the same buffer. The fraction of alpha-helix, F alpha (or beta-structure, F beta), increased with increasing PS (or PA) concentration, reaching a saturation value of about 0.7 (or about 1). Mixed vesicles comprising PS and dilauroylphosphatidylcholine (DLPC) also induced alpha-helix conformation, however, the saturation value of F alpha diminished with decreasing PS content in mixed vesicles. On the other hand, the spectral patterns for poly(L-lysine) in DMPG vesicle suspensions exhibited the coexistence of alpha-helix and beta-structure. Both F alpha and F beta increased with DMPG concentration and reached saturation values of about 0.5. Mixed vesicles composed of DMPG and dimyristoylphosphatidylcholine (DMPC) led to a reduction in F beta, while F alpha remained almost constant. The diversity in ordered structure induced by different phospholipid vesicles suggests the participation of lipid head groups in determining the secondary structure of poly(L-lysine) adsorbed on the vesicular surface.     

Involvement of prostaglandin-induced proteins in the inhibition of herpes simplex virus replication

Cyclopentenone prostaglandin (PG), delta7-PGA1 was found to induce several polypeptides in human embryonic fibroblast (HEF) cells which were noticed to be dose-related and appeared after 1 h of treatment with a peak at around 5 h and gradual disappearance after 12 h. PG-induced proteins were almost identical in terms of molecular weights with those induced by heat-shock at 42 degrees C. Regarding the mechanism of inhibition of herpes simplex virus (HSV) replication by PG in cell culture, dot blot hybridization has revealed that the level of immediate early (IE) mRNA of the virus was reduced after PG treatment with time dependence. And this delayed inhibitory effect of delta7-PGA1 on HSV was shown to be associated with the production and accumulation of the induced polypeptides.     

Interaction of surfactants with bilayer of negatively charged lipid: effect on gel-to-liquid-crystalline phase transition of dilauroylphosphatidic acid vesicle membrane

The interaction of surfactants with the vesicle membrane of the negatively charged lipid, dilauroylphosphatidic acid, was investigated through their effect on the gel-to-liquid-crystalline phase transition of the lipid bilayer. Three types of surfactants (anionic, cationic and non-ionic) with different hydrocarbon chain length were examined. (i) Anionic sodium alkylsulfates affected the phase transition temperature, Tm, only weakly. (ii) Non-ionic alkanoyl-N-methylglucamides decreased Tm monotonously with increasing concentration. The depression of Tm induced by these surfactants was analyzed by applying the van't Hoff model for the freezing-point depression, and the partition coefficients of the surfactants between bulk water and lipid membrane were estimated. (iii) Cationic alkyltrimethylammonium bromides affected Tm in a complex manner depending on the hydrocarbon chain length of the surfactants. Octyl-/tetradecyl-trimethylammonium bromide depressed/elevated Tm monotonously with increasing concentration, whereas the change in Tm induced by decyl- and dodecyltrimethylammonium bromides was not monotonous but biphasic. This complex behavior of the phase transition temperature was well explained, based on the statistical mechanical theory presented by Suezaki et al. (Biochim. Biophys. Acta, 818 (1985) 31-37), which takes into account the interaction between surfactant molecules incorporated in the lipid membrane.     

delta 12-Prostaglandin J2, an ultimate metabolite of prostaglandin D2 exerting cell growth inhibition

L-1210 murine leukemia cells were exposed to prostaglandin D2 (PGD2), 10 micrograms/ml, in culture medium for various time, and subsequent cell growth was observed. More than 24 h exposure to PGD2 was required to inhibit cell growth almost completely. During this period, PGD2 degraded time-dependently into several products. The major product was identified as delta 12-PGJ2 by TLC, UV and mass spectra. When delta 12-PGJ2 was added to cells instead of PGD2, it evoked growth inhibition with much shorter contact time than PGD2. In addition, when the medium containing PGD2 was preincubated at 37 degrees C for 24 h, it elicited growth inhibition with only 6 h contact with cells. Furthermore, when the medium containing PGD2 was changed every 6 h during 24 h exposure time to cells, no significant growth inhibition was observed. These results suggested that PGD2 per se has little, if any, growth inhibitory activity, and delta 12-PGJ2 is an ultimate metabolite exerting growth inhibition. This action appears to be independent of cAMP, since delta 12-PGJ2 was virtually inactive in raising intracellular cAMP levels.     

Structural organization of the human kininogen gene and a model for its evolution

The entire human kininogen gene has been isolated as a set of overlapping genomic DNA fragments, and the 11 exons encompassing approximately 27 kilobase pairs have been mapped by restriction enzyme analysis and nucleotide sequence determination. The nine 5'-terminal exons encode the 5'-untranslated region and the protein-coding region for the signal peptide and the heavy chain, which are common for high molecular weight (HMW) and low molecular weight (LMW) prekininogen mRNAs. Exon 10 consists of the common sequence for bradykinin and the immediately following unique sequence for HMW prekininogen mRNA. Exon 11 is then located following a 90-nucleotide sequence downstream from exon 10 and precisely specifies the sequence unique to LMW prekininogen mRNA. This, together with the hybridization analysis of total human cellular DNA, leads us to conclude that human HMW and LMW prekininogen mRNAs are produced from a single gene as a consequence of alternative RNA processing events. The structural analysis of the kininogen gene also shows that each of the nine 5'-terminal exons discretely specifies the nine protein domains observed in the amino-terminal portion of the kininogens. Furthermore, these nine genetic domains can be characterized by a thrice repeated pattern of three genetic segments, and two sets of these three domains, encompassing exons 3-5 and exons 6-8, are most closely related to each other. Therefore, we have proposed two successive duplication mechanisms as a model for the generation of the structure of the kininogen gene.